Project Summary/Abstract In recent years there has been a dramatic rise in antimicrobial drug resistance, with increasing concern regarding certain types of Gram-negative pathogens. Given that most of the attention in the natural products research community has been paid to Gram-positive organisms such as Streptomyces, which provide the majority of natural antibiotics that primarily target Gram-positive pathogens, the development of new genetic tools tailored towards manipulating biosynthetic pathways from Gram-negative bacteria represents an opportunity to access new anti-infective agents. Further, study of the regulation and resistance-mechanisms associated with antibiotic biosynthesis in Gram-negative bacteria may help us understand how drug resistance evolves and how to combat it. In light of these circumstances, the goal of this proposal is to develop new genetic tools and methods to access anti-infective agents from Gram-negative organisms and test their activity. I hypothesize that, following the development of these tools, I will be able to clone, express, and engineer the biosynthetic gene clusters responsible for producing the didemnins and thalassospiramides, potent antiviral and immunosuppressive peptides, respectively, from the Alphaproteobacterium Tistrella mobilis. Ultimately, this will enable generation of new analogues with potentially enhanced or altered bioactivity. To achieve this, I will first build a new, optimized bacterial artificial chromosome vector for targeted cloning of gene clusters. This will facilitate efficient cloning and stable maintenance of the large didemnin (did) and thalassospiramide (ttm) pathways that encode complex type I hybrid nonribosomal peptide synthetase- polyketide synthase (NRPS-PKS) enzymatic machines. I will integrate these captured pathways into the genomes of two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, for stable heterologous expression. Finally, I plan to develop tools and methods to combine CRISPR-cas9 and recombineering in E. coli to perform precise, multiplexed, and markerless editing of captured gene clusters. Although there have been many reports using CRISPR-cas9 to edit bacterial genomes, there has been no system developed for systematic editing of self-replicating plasmids in E. coli. Such a system would fit nicely into the current platform in the Moore lab to study gene clusters through cloning and heterologous expression. New analogues generated through this approach will be tested using bacterial cytological profiling, pioneered by our collaborator at UC San Diego, Professor Kit Pogliano, who is also my project co-sponsor. This proposal is designed to supplement my prior research experience and provide me with new technical and intellectual training opportunities through interactions with my Ph.D. adviser, Professor Moore, and project co-sponsor, Professor Kit Pogliano, who both have extensive experience advising predoctoral students. My mentors have been carefully chosen for their diverse and complementary scientific expertise to cover all elements of the proposed research.